title: Novel interaction of ornithine decarboxylase with sepiapterin reductase regulates neuroblastoma cell proliferation.
abstract: Ornithine decarboxylase (ODC) is the sentinel enzyme in polyamine biosynthesis. Both ODC and polyamines regulate cell division, proliferation, and apoptosis. Sepiapterin reductase (SPR) catalyzes the last step in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase, and has been implicated in neurological diseases but not yet in cancer. In this study, we present compelling evidence that native ODC and SPR physically interact, and we defined the individual amino acid residues involved in both enzymes using in silico protein-protein docking simulations. The resulting heterocomplex is a surprisingly compact structure, featuring two energetically and structurally equivalent binding modes both in monomer and in dimer conformations. The novel interaction between ODC and SPR proteins was confirmed under physiological conditions by co-immunoprecipitation and co-localization in neuroblastoma (NB) cells. Importantly, we showed that siRNA (small interfering RNA)-mediated knockdown of SPR expression significantly reduced endogenous ODC enzyme activity in NB cells, thus demonstrating the biological relevance of the ODC-SPR interaction. Finally, in a cohort of 88 human NB tumors, we found that high SPR mRNA expression correlated significantly with poor survival prognosis using a Kaplan-Meier analysis (log-rank test, P=5 × 10(-4)), suggesting an oncogenic role for SPR in NB tumorigenesis. In conclusion, we showed that ODC binds SPR and thus propose a new concept in which two well-characterized biochemical pathways converge via the interaction of two enzymes. We identified SPR as a novel regulator of ODC enzyme activity and, based on clinical evidence, present a model in which SPR drives ODC-mediated malignant progression in NB.
authors.affiliation: Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, USA.
authors.forename: Ingo
authors.initials: I
authors.lastname: Lange
authors.affiliation: Department of Pediatric Oncology/Hematology, Sophia Children's Hospital, Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands.
authors.forename: Dirk
authors.initials: D
authors.lastname: Geerts
authors.affiliation: Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Feith
authors.affiliation: Pacific Biosciences Research Center, University of Hawaii at Manoa, Honolulu, HI 96822, USA.
authors.forename: Gabor
authors.initials: G
authors.lastname: Mocz
authors.affiliation: Department of Oncogenomics, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: Koster
authors.affiliation: Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, USA; Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA. Electronic address: andre@hawaii.edu.
authors.forename: André S
authors.initials: AS
authors.lastname: Bachmann
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D006801:Humans; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009447:Neuroblastoma / Q000401:mortality / Q000473:pathology; D009474:Neurons / Q000502:physiology*; D009955:Ornithine Decarboxylase / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D016019:Survival Analysis
chemical_list: D000429:Alcohol Oxidoreductases; C022630:sepiapterin reductase; D009955:Ornithine Decarboxylase
keywords: 4′,6-diamidino-2-phenylindole; BP; DAPI; FBS; HEK293; NB; NOS; ODC; PBS; SPR; blocking peptide; docking simulation; fetal bovine serum; human embryonic kidney 293; neuroblastoma; nitric oxide synthase; ornithine decarboxylase; pediatric neuroblastoma; phosphate-buffered saline; protein–protein interaction; sepiapterin reductase